Screening Of Bacillus Strains Against Rice Blast And Research Of Biocontrol Mechanism

Rice blast, one of the most devastating diseases of cultivated rice, affects rice industry in sustainable development and a constant threat to global food security. Chemical pesticides and disease-resistant varieties potentially effective control rice blast in present. Because the physiological race of Magnaporthe oryzae changes easily and the breeding time for cultivar-resistance is long. Especially, chemicals yet pose residual toxicity and environmental pollution. Therefore, it had a vital significance for crop safety to finding an effective way to biological control rice blast. The antifungal and growth-promoting properties of Bacillus species indicate the potential role as biological control of the disease. This study aimed to screen Bacillus strains to control rice blast and evaluating the biocontrol efficacy of experimental strains in vitro determination of blade, under greenhouse condition and in field. The study also determined its classification status and analysis the biocontrol mechanism of Bacillus strains effectively against rice blast.The total 280 bacteria isolated from rice leave surface, only three isolates had highly antifungal efficacy through experimental screening, such as strains SYX04, SYX20 and S-22. The results of 2-years greenhouse and field experiments carried out in 5 regions in Liaoning and Fujian province indicated that the efficacy of strain SYX04 controlled leaf blast by 71.5%～84.4% and panicle blast by 63.5%～83.8%. The efficacy of strain SYX20 controlled leaf blast by 73.5%～85.1% and panicle blast by 64.0%～85.6%. The efficacy of strain S-22 controlled rice leaf blast by 69.2%～78.3% and panicle blast by 61.6%～77.9%. The experimental results showed that strains SYX04, SYX20 and S-22 played a good role in rice growth, such as promoting rice root length, increasing rice length, panicle weight and thousand seed weight. By the comparing of 16S rDNA sequences, combined with its results of morphological characteristic and biolog analysis, strains SYX04 and SYX20 were identified as B. subtilis and strain S-22 was identified as S. multivorum.The antifungal efficacy of B. subtilis SYX04 and SYX20 culture inhibiting spore germination, germ tube and appressorial formation were up to 88.3%, it of culture filtrate being up to 70%, and inhibiting efficacy of crude extraction deposited by 80%(NH4)2SO4 being about 70%. Strains SYX04 resulted in shrinkage and plasmolysis of hyphae, the cell wall of conidia degraded severely and abnormal infection hyphae formed. Strains SYX20 resulted in swelling and partial distortion of hyphae, cell wall of hyphae and conidia degraded severely and protoplasm leaked, and abnormal infection hyphae formed. The antifungal efficacy of S. multivorum S-22 culture and filtrate were 65～70%. Strain S-22 resulted in constriction of hyphae, the cell wall perforation of conidia and abnormal infection hyphae formed. All test bacteria could form biofilm and produce some relevant biocontrol material, such as protease, amylase and cellulose. Peroxidase (PO), Polyphenol oxidase (PPO) and superoxide dismutase (SOD) associated with disease resistance characteristics were induced in rice plants after exposure to B. subtilis SYX04 and SYX20 so as to induce defense response.All test bacteria could improve soil nutrition status of rice root. The colonization in rice plant of B. subtilis SYX04 and SYX20 was studied through GFP-tagged cell. The root-irrigation results indicated that strains SYX04 and SYX20 could colonize stablely in epidermis, exodermis, cortex parenchyma cells, endodermis, pericycle and the primary microtubule organization in rice roots; epidermis, ground tissue and vascular bundle in rice stems. The results of foliage spray indicated that strains SYX04 and SYX20 had a strong ability to colonize in epidermal hair, epidermis, stoma, parenchymal cell, sclerenchyma, bulliform cell and vascular bundle in rice leave. More than 5×106CFU/g labeled strains were detected after 20 days in rice mature leaves and more than 1 x 105 CFU/g labeled strains could be also detected in rice newly grown leaves.Therefore, B.subtilis SYX20 and B.subtilis SYX04 could be used as biocontrol agents to control rice blast in Southern and Northern regions of China. It has a guiding significance for the prevention of rice blast in rice sustainable development.